Hank s balanced salt solution

Umbilical cords from 4 foals were collected immediately after birth and 10 cm length of the umbilical cord was placed in a jar with PBS and 5% penicillin-streptomycin for transport. In the laboratory, the umbilical cord was first washed in 70% ethanol and then again in PBS. Next, the vein and Wharton's jelly tissue were removed and the umbilical arteries were carefully dissected. One artery was used to recover endothelial cells for co-culture experiments as described below, and the other artery was subjected to an arterial ring assay as described in Supplementary material 1. In order to obtain the endothelial cells, the respective umbilical artery was filled with collagenase I [Gibco™, ThermoFisher Scientific; 0.8 mg/ml in Hank's Balanced Salt Solution (HyClone Laboratories, Logan, Utah, USA)] and the ends were closed with clamps. Then the umbilical artery was incubated for 30 min at 37 °C and massaged twice during this time. After the incubation period, the content of the artery was transferred to a centrifuge tube and the artery was massaged again while washed with PBS, which was also transferred to the same centrifuge tube thereafter. The isolated cells were pelleted (390 x g, 5 min), washed with PBS and seeded in tissue-culture-coated flasks in FBS-supplemented medium until cryopreservation in liquid nitrogen.

Placental chorionic villi were dissected within 1–2 h following delivery and washed 3 times with gentle shaking using Hanks’ Balanced Salt Solution (Hyclone Laboratories Inc, ThermoFisher Scientific, Waltham, MA, USA). A self-regenerating explant culture method was adapted from Siman et al. and Trowell [80 (link),81 (link)] by culturing explants at the gas-liquid interface. Briefly, three 2 mm blocks of chorionic villi were placed onto inserts with 8 μm pore polyethylene terephthalate membranes (353182, ThermoFisher Scientific, Waltham, MA, USA). Inserts were placed into wells in a companion 12-well plate. 550 µL of Iscove’s Modified Dulbecco’s Media (IMDM, Hyclone Laboratories Inc, ThermoFisher Scientific, Waltham, MA, USA) with 1% Penn/Strep, 1% antibacterial/antimycotic, and 1% ITS + 1 Liquid Media Supplement (MilliporeSigma, Toronto, ON, Canada) was added to the wells in the companion plates. The plates were incubated at 37°C in 5% CO₂ and the media was changed every 48 h.